10.3.1. Introduction

Multiplex Ligation-dependent Probe
Amplification (MLPA) technology is an amplification technique that allows
simultaneous detection of up to 40 targets with the use of a single PCR primer
pair. The procedure uses a series of paired oligonucleotides (half-probes),
each pair specific for one target. The two half-probes; the Left Probe Oligo
(LPO) and the Right Probe Oligo (RPO) lie adjacent to each other on the target
genome so that they can be joined together by a ligation reaction, to produce
an amplification probe (Fig. 6). In addition to a target-specific sequence,
each of the half-probes contains one of two sequences recognized by a universal
PCR primer, for probe amplification. Since these PCR primer sequences are
common to all half-probe pairs, a single pair of PCR primers can amplify all
target probes in a multiplex reaction. The half-probe pairs also contain a
‘stuffer’ fragment of variable length, allowing each amplified probe to be
identified by its size, using (capillary) electrophoresis (Fig. 6). This
technique was recently adopted to detect the most common honey bee viruses
including CBPV, DWV (KV & VDV-1), ABPV (IAPV & KBV), BQCV, SBPV, SBV
(De Smet et al., 2012). Because these
are all RNA viruses, the MLPA is preceded by a reverse transcription of RNA
into cDNA. Since the probes are strand-specific, this technique is highly
suitable for the selective detection of either the positive-strand genomic
viral RNA or the negative-strand virus replicative intermediate RNA, which is a
marker for virus replication.

Since
several targets are amplified at the same time, there will be competition
between different targets for the amplification resources (primers,
nucleotides, enzyme). This ‘competitive’ PCR allows for a measure of relative
quantification between the targets, in the sense that the relative proportion
of the targets after amplification should, if all targets amplify equally
efficiently, reflect their initial proportions in the sample. By including one
or more internal reference genes or exogenously added absolute quantification
standards among the targets, the procedure can be made (semi- quantitative.